The discovery of β-lactam antibiotics was one of the most important steps in the struggle against pathogenic bacteria. β-Lactam antibiotics such as penicillins and cephalosporins are one of the three largest antibiotic classes and the most heavily prescribed antibiotics in clinical use today. They target enzymes that synthesize the bacterial cell wall. However, increased resistance of bacterial infections to antibiotic treatment has been extensively documented and has become a generally recognized problem for clinicians worldwide, in both hospital and community settings (see, e.g., Levy, S. B. Scientific American (1998) 278, 3, 46-53; Fisher, J. F., et al. Chem. Rev. (2005) 105, 395-424). One mechanism of bacterial self-defense against β-lactam antibiotics is the production of β-lactamases (Bla), bacterial enzymes that can hydrolyze the β-lactam ring in penicillins and cephalosporins with high catalytic efficiency and render the bacteria resistant to the β-lactam antimicrobial reagents (see, e.g., Wilke, M. S., et al. Current Opinion in Microbiology (2005) 8, 525-533).
β-Lactamases are organized into four molecular classes (A, B, C and D) based on their amino acid sequence, their substrate spectrum and responses to inhibitors. Class A enzymes have a molecular weight of about 29 kDa and can preferentially hydrolyze penicillins. Class B enzymes include metalloenzymes that have a broader substrates profile than other β-lactamase classes. Class C enzymes have molecular weights of approximately 39 kDa and include the chromosomal cephalosporinases of gram-negative bacteria, which are responsible for the resistance of gram-negative bacteria to a variety of both traditional and newly designed antibiotics. In addition, class C enzymes also include the lactamase of P99 Enterobacter cloacae, which is responsible for making this Enterobacter species one of the most widely spread bacterial agents in United States hospitals. The recently recognized class D enzymes are serine hydrolases, which exhibit a unique substrates profile. The spread of antibiotics resistance conferred by expression of β-lactamase in bacteria threatens the ability to treat bacterial infections.
Therefore, detecting β-lactamases and screening their inhibitors (see, e.g., Buynak, J. D., Biochemical Pharmacology (2006) 71, 930-940) in biological samples before conducting the efficient antibiotic therapy, is extremely important clinically. Accordingly procedures for detecting β-lactamases have been developed such as fluorescent (e.g., genotyping based on polymerase chain reaction (PCR)) or chromogenic assays (such as the well known nitrocefin and PADAC indicators). Some other fluorogenic and hydrogel based substrates have also been developed as reporters for imaging the gene expression of β-lactamases in vitro and in vivo (Zlokarnik, L., et al., Science (1998) 279, 84-88; Gao, W. Z., et al., J. Am. Chem. Soc. (2003) 125, 11146-11147; Xing, B. G., et al., J. Am. Chem. Soc. (2005) 127, 4158-4159; Yang, Z. M., et al., J. Am. Chem. Soc. (2007) 129, 266-267).
However, current detection methods have significant drawbacks such as laborious manipulation, time-consuming processes, requirement for highly specific instrumentation, and limited chemical stability and aqueous solubility of reagents used. There is therefore a need for a simple, rapid, sensitive and economical detection method.
Accordingly, it is an object of the present invention to provide an alternative method of detecting β-lactamases that avoids these disadvantages.
This object is solved by modulating the formation of such complex among others by the methods as described in the independent claims.